Mary Jo Hamilton

Suppression of proliferative response of BoCD4+ T lymphocytes by activated BoCD8+T lymphocytes in the mammary gland of cows with Staphylococcus aureus mastitis

Investigations were conducted to determine the mechanisms that account for differences in the responses of BoCD4+ lymphocytes from mammary gland secretions (MGS) in healthy cows and in cows with Staphylococcus aureus infection. The proliferative response to lectins and S. aureus antigens of mammary gland lymphocytes from healthy, S. aureus immunized cows was less than the response of peripheral blood lymphocytes. The lower responses of mammary gland lymphocytes were attributable both to less efficient antigen presentation by mammary gland antigen-presenting cells (APC) than by peripheral blood APC, and to lower responsiveness of mammary gland lymphocytes to lectins and antigen. In addition, the proliferative response of infected mammary gland lymphocytes was less than the response of uninfected mammary gland lymphocytes. This difference resulted from decreased proliferation of BoCD4+ lymphocytes in infected MGS. Flow cytometric analysis revealed that infected MGS contained increased numbers of BoCD8+ cells which coexpressed an activation molecule, ACT2, relative to BoCD8+ cells from uninfected MGS. Removal of BoCD8+, ACT2+ lymphocytes resulted in increased antigen responsiveness by lymphocytes from infected mammary glands. Also, when purified BoCD4+ lymphocytes were stimulated with antigen in the presence of varying numbers of ACT2+, BoCD8+ lymphocytes, antigen responsiveness was decreased in a dose-related manner. These data demonstrate that hyporesponsiveness of mammary gland lymphocytes to lectins and S. aureus antigen is, in part, mediated by activated BoCD8+ lymphocytes and suggest that this population enhances persistent intramammary infection by S. aureus.

Report of the Second Equine Leucocyte Antigen Workshop, Squaw Valley, California, July 1995

The final assignment of antibody clusters for leucocyte antigens and immunoglobulins, as described in detail in Sections 3 and 4, is summarized in Table 4. Together with other mAbs developed outside of ELAW II (Table 9) this pool of reagents represent a powerful array of tools for the study of equine immunity. The Second Equine Leucocyte Antigen Workshop made considerable advances in pursuing the objectives of establishing the specificities of mAbs and achieving consensus on the nomenclature for equine leucocyte and immunoglobulin molecules. Of equal importance, several productive collaborations were fostered among the participating laboratories and observers. Overall, enormous advances have been made in the past decade since mAbs specific for equine leucocyte antigens and immunoglobulins were first reported. There remains enormous scope and need for further studies of equine leucocyte antigens and immunoglobulins, both for the purposes of comparative immunology and for the good of the horse. In the future novel techniques will be required to develop reagents for specific target antigens such as the orthologues of the CD25 or CD45 isoforms. In studies of equine immunoglobulins the functional role of the IgG isotypes must be better established, reagents for IgE must be developed, and cloning of the immunoglobulin heavy chain genes will be essential if the complexities of the IgG sub-isotypes are to be elucidated. The tasks still facing the currently small group of equine immunologists throughout the world remain formidable, and will only be tackled successfully in a spirit of collaboration.

Differential distribution of γδT-cell receptor lymphocyte subpopulations in blood and spleen of young and adult cattle

Abstract A panel of monoclonal antibodies to bovine leukocyte differentiation molecules was used to evaluate peripheral blood and splenic lymphocytes from cattle of various ages. The major population of peripheral blood lymphocytes from neonatal calves was γδT-cell receptor (TCR1) positive, as determined by TCR1-N12 expression. TCR1-N12 + lymphocytes were decreased in number in older calves, and were lowest in adult cattle. The major subpopulation of TCR1-N12 + cells from peripheral blood coexpressed WC1, but not BoCD2. A small subpopulation of peripheral blood TCR1-N12 + cells from cattle of all ages coexpressed BoCD2, but not WC1. The TCR1-N12 + BoCD2 + lymphocytes made up the largest TCR1-N12 + lymphocyte subpopulation in spleens of both calves and adults. The TCR1-N12 + WC1 + splenic lymphocytes were present as a small population. The data indicate that two subpopulations of TCR1 + lymphocytes are present in cattle of all ages. These two subpopulations are differentially distributed between blood and spleen, with TCR1-N12 + WC1 + lymphocytes predominating in blood, and TCR1-N12 + BoCD2 + cells predominating in spleen.

Identification of a monoclonal antibody reactive with the bovine orthologue of CD3 (BoCD3)

A monoclonal antibody (mAb), MM1A, that identifies a molecule expressed on a large percentage of bovine lymphocytes (60-80%) was examined to determine its specificity. Two and three colour immunofluorescence analysis using flow cytometry revealed the molecule is highly expressed on all CD4+ and CD8+ lymphocytes and lymphocytes that express WC1 and the gamma/delta TCR. In contrast to mAbs reactive with BoCD5, MM1A did not react with B lymphocytes. Biochemical analysis revealed that the mAb immunoprecipitates a molecular complex comprising a set of peptides with M(r) approximately 12, 16, 22, 32, 36, and 44 kDa under reducing conditions and an additional 96 kDa peptide complex under non-reducing conditions. The data indicate that MM1A recognizes the bovine orthologue of CD3 (BoCD3).

Clearance of Virulent but Not Avirulent Rhodococcus equi from the Lungs of Adult Horses Is Associated with Intracytoplasmic Gamma Interferon Production by CD4+ and CD8+ T Lymphocytes

ABSTRACT Rhodococcus equi is a gram-positive bacterium that infects alveolar macrophages and causes rhodococcal pneumonia in horses and humans. The virulence plasmid of R. equi appears to be required for both pathogenicity in the horse and the induction of protective immunity. An understanding of the mechanisms by which virulent R. equi circumvents protective host responses and by which bacteria are ultimately cleared is important for development of an effective vaccine. Six adult horses were challenged with either virulent R. equi or an avirulent, plasmid-cured derivative. By using a flow cytometric method for intracytoplasmic detection of gamma interferon (IFN-γ) in equine bronchoalveolar lavage fluid (BALF) cells, clearance of the virulent strain was shown to be associated with increased numbers of pulmonary CD4+ and CD8+ T lymphocytes producing IFN-γ. There was no change in IFN-γ-positive cells in peripheral blood, suggesting that a type 1 recall response at the site of challenge was protective. The plasmid-cured strain of R. equi was cleared in horses without a significant increase in IFN-γ-producing T lymphocytes in BALF. In contrast to these data, a previous report in foals suggested an immunomodulating role for R. equi virulence plasmid-encoded products in downregulating IFN-γ expression by equine CD4+ T lymphocytes. Intracytoplasmic detection of IFN-γ provides a method to better determine whether modulation of macrophage-activating cytokines by virulent strains occurs uniquely in neonates and contributes to their susceptibility to rhodococcal pneumonia.

Invasion and Persistence of Mycobacterium avium subsp. paratuberculosis during Early Stages of Johne's Disease in Calves

ABSTRACT Infection with Mycobacterium avium subsp. paratuberculosis causes Johnes disease in cattle and is a serious problem for the dairy industry worldwide. Development of models to mimic aspects of Johnes disease remains an elusive goal because of the chronic nature of the disease. In this report, we describe a surgical approach employed to characterize the very early stages of infection of calves with M. avium subsp. paratuberculosis. To our surprise, strains of M. avium subsp. paratuberculosis were able to traverse the intestinal tissues within 1 h of infection in order to colonize distant organs, such as the liver and lymph nodes. Both the ileum and the mesenteric lymph nodes were persistently infected for months following intestinal deposition of M. avium subsp. paratuberculosis despite a lack of fecal shedding of mycobacteria. During the first 9 months of infection, humoral immune responses were not detected. Nonetheless, using flow cytometric analysis, we detected a significant change in the cells participating in the inflammatory responses of infected calves compared to cells in a control animal. Additionally, the levels of cytokines detected in both the ileum and the lymph nodes indicated that there were TH1-type-associated cellular responses but not TH2-type-associated humoral responses. Finally, surgical inoculation of a wild-type strain and a mutant M. avium subsp. paratuberculosis strain (with an inactivated gcpE gene) demonstrated the ability of the model which we developed to differentiate between the wild-type strain and a mutant strain of M. avium subsp. paratuberculosis deficient in tissue colonization and invasion. Overall, novel insights into the early stages of Johnes disease were obtained, and a practical model of mycobacterial invasiveness was developed. A similar approach can be used for other enteric bacteria.

Analysis of the Immune Response to Mycobacterium avium subsp. paratuberculosis in Experimentally Infected Calves

ABSTRACT Johnes disease of cattle is widespread and causes significant economic loss to producers. Control has been hindered by limited understanding of the immune response to the causative agent, Mycobacterium avium subsp. paratuberculosis, and lack of an effective vaccine and sensitive specific diagnostic assays. The present study was conducted to gain insight into factors affecting the immune response to M. avium subsp. paratuberculosis. A persistent proliferative response to M. avium subsp. paratuberculosis purified protein derivative and soluble M. avium subsp. paratuberculosis antigens was detected in orally infected neonatal calves 6 months postinfection (p.i.) by flow cytometry (FC). CD4+ T cells with a memory phenotype (CD45R0+) expressing CD25 and CD26 were the predominant cell type responding to antigens. Few CD8+ T cells proliferated in response to antigens until 18 months p.i. γδ T cells did not appear to respond to antigen until 18 months p.i. The majority of WC1+ CD2− and a few WC1− CD2+ γδ T cells expressed CD25 at time zero. By 18 months, however, subsets of γδ T cells from both control and infected animals showed an increase in expression of CD25, ACT2, and CD26 in the presence of the antigens. Two populations of CD3− non-T non-B null cells, CD2+ and CD2−, proliferated in cell cultures from some control and infected animals during the study, with and without antigen. The studies clearly show multicolor FC offers a consistent reliable way to monitor the evolution and changes in the immune response to M. avium subsp. paratuberculosis that occur during disease progression.

Multiplex immunoassay for serological diagnosis of Mycobacterium bovis infection in cattle.

ABSTRACT Efforts to develop a better diagnostic assay for bovine tuberculosis have shown that the sensitivity and specificity of an assay can be improved by the use of two or more antigens. As reported here, we developed a multiplex chemiluminescent immunoassay that can simultaneously detect antibody activity to 25 antigens in a single well in a 96-well plate array format. The chemiluminescent signal is captured with a digital imaging system and analyzed with a macro program that tracks each serum for its pattern of antibody activity for Mycobacterium bovis antigens. The comparison of sera from 522 infected and 1,489 uninfected animals showed that a sensitivity of 93.1% and a specificity of 98.4% can be achieved with a combination of antigens. The assay system is rapid and can be automated for use in a centralized laboratory.

Temporal Patterns of Diagnostic Results in Serial Samples from Cattle with Advanced Paratuberculosis Infections

As part of investigating diagnostic strategies for Mycobacterium avium subsp. paratuberculosis (Map), serial results from polymerase chain reaction (PCR) on extraintestinal tissues (blood, milk, and liver) were compared with those from more conventional detection methods including serum enzyme-linked immunosorbent assay (ELISA), fecal culture, and fecal PCR. Three cows previously identified as being subclinically infected with Map were selected for the study. Blood, milk, and feces were collected daily and liver biopsies were obtained weekly for a 30-day period. Unexpectedly, a substantial daily variation in serum ELISA sample to positive (S/P) ratios was observed in all 3 cows. In contrast, fecal culture results were consistently positive. However, whereas fecal culture colony counts were consistently high for 2 cows throughout the study, colony counts from the third cow varied from day to day. Diagnostic sensitivity of PCR for fecal, blood, milk, and liver samples in these advanced subclinically infected cows was 87%, 40%, 96%, and 93%, respectively.

Evaluation of two mutants of Mycobacterium avium subsp. paratuberculosis as candidates for a live attenuated vaccine for Johne's disease.

Control of Johnes disease, caused by Mycobacterium avium subsp. paratuberculosis, has been difficult because of a lack of an effective vaccine. To address this problem we used targeted gene disruption to develop candidate mutants with impaired capacity to survive ex vivo and in vivo to test as a vaccine. We selected relA and pknG, genes known to be important virulence factors in Mycobacterium tuberculosis and Mycobacterium bovis, for initial studies. Deletion mutants were made in a wild type Map (K10) and its recombinant strain expressing the green fluorescent protein (K10-GFP). Comparison of survival in an ex vivo assay revealed deletion of either gene attenuated survival in monocyte-derived macrophages compared to survival of wild-type K10. In contrast, study in calves revealed survival in vivo was mainly affected by deletion of relA. Bacteria were detected in tissues from wild-type and the pknG mutant infected calves by bacterial culture and PCR at three months post infection. No bacteria were detected in tissues from calves infected with the relA mutant (P<0.05). Flow cytometric analysis of the immune response to the wild-type K10-GFP and the mutant strains showed deletion of either gene did not affect their capacity to elicit a strong proliferative response to soluble antigen extract or live Map. Quantitative RT-PCR revealed genes encoding IFN-γ, IL-17, IL-22, T-bet, RORC, and granulysin were up-regulated in PBMC stimulated with live Map three months post infection compared to the response of PBMC pre-infection. A challenge study in kid goats showed deletion of pknG did not interfere with establishment of an infection. As in calves, deletion of relA attenuated survival in vivo. The mutant also elicited an immune response that limited colonization by challenge wild type Map. The findings show the relA mutant is a good candidate for development of a live attenuated vaccine for Johnes disease.